UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

AGRICULTURAL   SCIENCES 

Vol.  1,  No.  7,  pp.  141-172  April  25,  1914 


STUDIES  ON  AMMONIFICATION  IN  SOILS 
BY  PURE  CULTURES 

BY 

C.  B.  LIPMAN  and  P.  S.  BURGESS 


The  study  of  the  physiological  efficiency  of  soil  bacteria 
rather  than  their  number  is  admittedly  the  dominant  method  in 
soil  bacteriological  investigations.  In  view  of  this  fact  it  is 
singularly  striking  to  note  how  little  work  has  been  accomplished 
in  the  study  of  some  phases  of  the  physiological  efficiency  of  pure 
cultures  of  certain  groups  of  soil  bacteria.  This  is  especially  so, 
since  the  introduction  of  radical  changes  in  our  soil  bacterio- 
logical methods  have  made  it  necessary  to  repeat  some,  if  not  all, 
of  the  work  which  had  been  carried  out  by  the  old  methods. 
However  this  may  be,  it  remains  a  fact  that,  since  the  publication 
of  MarchalV  splendid  work  on  ammonification  in  solution 
cultures,  but  scant  information  has  been  adduced  from  studies 
of  soil  bacteria  which  relate  to  the  physiology  as  well  as  the 
physiological  efficiency  of  even  the  more  common  ammonifying 
bacteria.  Certainly,  the  work  carried  out  along  this  line  in 
direct  soil  cultures,  which  recent  work  has  shown  to  be  so  far 
superior  to  solution  cultures,  has  been  very  meager  indeed. 

In  order,  therefore,  to  glean  some  useful  information  relative 
to  the  physiological  efficiency  of  pure  cultures  of  a  number  of 
ammonia-producing  bacteria,  the  writers  deemed  it  wise  to  select 
a  number  of  organisms  and  to  compare  their  power  to  produce 
ammonia  not  only  from  one  form  of  organic  matter,  but  from 
several  forms,  most  of  which  have  found  use  in  farm  prac- 
tice as  fertilizers.    Accordingly,  the  following  organisms  in  pure 


142        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

culture  were  selected  for  the  experiments :  B.  mesentericus  vul- 
gatus,  Ps.  putida,  B.  vulgatus,  B.  megatherium,  B.  mycoides,  B. 
sub t His,  B.  tumescens,  Sarcina  lutea,  B.  proteus  vulgaris,  B. 
icteroides,  B.  ramosus,  Streptothrix,  sp.,  Ps.  fluorescens,  B.  vul- 
garis (Novy  strain),  Mic.  tetragenus.  The  organic  materials 
experimented  with  were  dried  blood  (13.16%  N),  tankage 
(9.62%  N),  cottonseed  meal  (5.5%  N),  sheep  and  goat  manure 
(2.13%  N),  peptone  (14.14%  N),  fish  guano  (8.63%  N),  and  bat 
guano  (3.96%  N). 

Method  of  Experiments 

Fifty-gram  portions  of  soil  were  placed  in  tumblers  and 
thoroughly  mixed  with  the  organic  material  to  be  tested.  The 
tumblers  were  covered  with  Petri  dish  covers  and  sterilized  in 
the  antoclave  at  a  pressure  of  thirty  pounds  for  three  hours. 
After  cooling,  the  soils  were  each  inoculated  with  a  1  cc.  suspen- 
sion of  the  organisms  to  be  tested,  made  up  by  shaking  with  some 
sterile  water  a  young  slope  culture  grown  on  bouillon  agar. 
The  soil  was  then  stirred  with  a  sterile  spatula  after  enough 
sterile  water  had  been  added  to  make  a  moisture  content  in  the 
soil  about  equal  to  the  optimum.  The  soil  cultures  thus  pre- 
pared were  incubated  at  28°  to  30°  C  for  twelve  days.  After 
the  incubation  period  the  soils  were  transferred  to  copper  dis- 
tilling flasks,  400  cc.  of  distilled  water  and  an  excess  of  Mg  O 
added,  and  distilled  into  standard  H/10  HC1.  The  ammonia 
was  then  determined  in  the  usual  way. 

No  attempt  was  made  to  run  all  the  series  with  the  different 
forms  or  organic  matter  at  the  same  time,  because  only  the 
relative  powers  of  the  different  organisms  to  produce  ammonia 
were  sought.  For  the  same  reason  amounts  of  organic  matter 
were  chosen  in  the  different  series  which  would  least  affect  the 
physical  conditions  obtaining  in  the  cultures  rather  than  amounts 
employed  which  would  make  the  total  amount  of  nitrogen  added 
the  same  in  all  scries. 

For  the  reasons  above  given,  therefore,  the  effects  of  the 
various  organisms  on  any  given  form  of  organic  material  will 
be  treated  below  as  a  separate  series  in  the  case  of  each  soil  and 


1914]        Lipman-Burgess :  Ammoniftcation  in  Sails  by  Pure  Cultures 


143 


comparisons  between  the  different  series  made  only  where  per- 
missible. The  data,  moreover,  are  presented  so  that  the  dupli- 
cate determinations  which  were  carried  out  in  all  cases  may  be 
compared.  The  averages  of  duplicate  determinations,  however, 
are  also  given  as  well  as  the  percentages  of  nitrogen  in  the 
organic  matter  which  was  transformed  to  ammonia. 

Three  different  California  soils  were  tested  with  each  one  of 
the  ammonifiable  materials.  The  soils  were  a  sandy  soil  from 
Anaheim,  a  clay  loam  from  Davis,  and  a  black  clay-adobe  soil 
from  Berkeley.  The  mechanical  and  chemical  analyses  of  these 
soils  are  given  in  Tables  I  and  II  which  follow. 


TABLE 

I 

Mechanical  Analyses  op  Soils 

Hyd.  value 

Sandy 

Clay-Loam 

Clay-Adobe 

Clay 

5.78 

19.12 

31.93 

0.25  mm. 

14.59 

40.93 

25.77 

0.50 

1.04 

3.35 

3.40 

1 

2.30 

6.60 

4.77 

2 

5.28 

7.75 

7.49 

4 

9.62 

8.78 

6.20 

8 

11.58 

8.10 

.87 

16 

4.87 

3.30 

2.78 

32 

15.23 

4.15 

7.66 

64 

29.40 

TABLE 

II 

3.07 

5.44 

Chemical  Analysis 

of  Soils 

Sandy 

Clay-Loam 

Clay-Adobe 

Insoluble  matter 

73.59 

53.55 

I  77.84 

Soluble 

silica 

11.17 

19.77 

K20 

.64 

.75 

.45 

Na20 

.15 

.11 

.07 

CaO 

1.39 

.82 

1.05 

MgO 

.93 

1.39 

1.21 

Mn304 

.04 

.04 

.08 

Fe203 

5.10 

7.56 

1.68 

A1203 

3.92 

10.04 

7.79 

PA 

.12 

.13 

.23 

S03 

.02 

.03 

.08 

Water  and 

organic 

matter 

2.88 

5.62 

5.72 

144        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

The  pure  cultures  of  the  organisms  employed  in  these  investi- 
gations were  obtained  by  one  of  us  from  the  bacteriological 
laboratories  of  the  University  of  Illinois.  Fresh  strains  of 
B.  subtilis  and  B.  mycoides  were,  however,  isolated  by  us  from 
California  soils  in  order  to  check  the  stock  cultures  of  the  same 
organisms.  The  same  relative  powers  of  producing  ammonia 
were,  however,  found  to  obtain  with  the  fresh  as  with  the  old, 
and  different  strains  thus  strengthening  the  validity  of  the 
results  below  discussed. 

Series  I.    Experiments  with  Dried  Blood 
Sandy  Soil 

Two  per  cent  of  finely  sifted  dried  blood  was  added  to  the 
soils  in  this  series,  or  one  gram  per  fifty  grams  of  soil.  In  the 
case  of  the  sandy  soil  three  series  in  duplicate  were  tried,  and  the 
same  relation  under  different  conditions  having  been  found  to 
obtain  between  the  ammonifying  powers  of  the  different  organ- 
isms, we  give  only  one  duplicate  set  of  the  determinations.  The 
incubation  period  was  twelve  days  throughout  at  a  temperature 
varying  between  27°  C  and  30°  C.  The  results  of  the  ammoni- 
fication  determinations  with  all  the  soils  using  dried  blood 
throughout  are  given  in  Table  III.  The  percentage  of  nitrogen 
of  the  total  amount  added  which  is  made  available  is  also  given 
in  every  case. 

The  data  in  Table  III  most  strikingly  indicate  the  superiority 
of  Ps.  putida,  B.  vulgaris  and  Sarcina  lutea  to  all  other  organisms 
in  their  efficiency  at  the  production  of  ammonia  from  the  nitrogen 
of  dried  blood.  The  next  fact  of  singular  interest  is  that  B. 
mycoides,  which  has,  in  the  hands  of  several  investigators,  shown 
such  marked  superiority  over  other  ammonifying  organisms  in 
the  production  of  ammonia  from  organic  nitrogen  in  solutions, 
manifests  a  relatively  low  power  to  transform  the  nitrogen  of 
dried  blood  in  soil  cultures  into  ammonia.  This,  moreover, 
cannot  be  considered  accidental,  since  different  strains  of  B. 
mycoides,  as  above  explained,  exhibited  that  same  lack  of  vigor  in 
three  duplicate  sets  of  cultures  run  at  different  times  and  under 
slightly  varying  conditions  of  temperature  and  period  of  incuba- 


1914]        Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures 


145 


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146        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

tion.  Of  the  three  organisms  showing  the  highest  efficiency,  as 
above  indicated,  in  transforming  the  nitrogen  of  dried  blood  to 
ammonia.  B.  vulgaris  appears  in  its  turn  to  be  the  most  vigorous, 
though  the  other  two  approach  it  closely  and  are  about  equal 
among  themselves. 

Clay-Loam  Soil 

"When  the  clay-loam  soil  is  used  as  the  medium  with  dried 
blood,  marked  differences  are  apparent  in  the  efficiency  of  all 
the  organisms.  The  poorer  air  supply  of  the  clay-loam  soil,  due 
to  its  fineness  and  much  greater  tenacity,  are  evidently  inimical 
to  ammonia  production,  even  though  the  same  source  of  nitrogen 
— dried  blood — is  supplied  for  the  ammonification  process.  The 
largest  amount  of  ammonia  produced  in  this  series  was  that  by 
B.  proteus  vulgaris,  and  even  that  was  little  more  than  one 
quarter  of  the  amount  produced  by  B.  vulgaris  as  above  noted 
in  the  sandy  soil. 

Moreover,  the  most  efficient  transformers  of  the  nitrogen  in 
dried  blood  in  the  sandy  soil  medium  are  not  necessarily  the 
same  as  those  in  the  clay-loam  soil.  For  example,  in  the  case  of 
the  clay-loam  soil  B.  proteus  vulgaris  is  the  most  efficient 
ammonia  producer  with  dried  blood  as  ammonifiable  material, 
while  in  the  sandy  soil  the  same  organism  manifests  less  than  half 
the  ammonifying  efficiency  of  B.  vulgaris.  This  latter  organism, 
however,  stands  second  to  B.  proteus  vulgaris  in  efficiency  in 
the  clay-loam  soil  and  yields  7.49  mgs.  of  ammonia  nitrogen  as 
against  9.10  mgs.  produced  by  the  last-named  organism.  There 
appear  to  be  four  other  organisms  which  approach  the  efficiency 
of  the  two  just  discussed  in  the  clay-loam  soil  in  the  following 
order:  Mic.  tetragenus,  B.  ramosus,  Streptothrix,  sp.,  and  Ps. 
putida.  While  the  latter  does  not  compare  in  efficiency  in  the 
clay-loam  soil  with  that  shown  by  it  in  the  sandy  soil,  it  still 
manifests  a  notable  efficiency.  Sarcina  lutea,  however,  appears 
to  have  lost  in  the  clay-loam  soil  the  marked  ammonifying  power 
possessed  by  it  in  the  sandy  soil. 

Of  course  il  must  be  again  emphasized  that  the  amounts  of 
ammonia    produced   by  all  the  organisms  in  the  clay-loam  soil 


1914]        Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures  147 

series  are  relatively  so  small  that  the  margin  allowable  for  safe 
comparison  must  of  necessity  be  much  decreased  and  therefore 
comparisons  are  more  difficult. 


Clay- Adobe  Soil 

Passing  on  to  a  study  of  the  data  obtained  with  the  clay-adobe 
soil  as  a  medium,  we  find  again  that  he  physical  condition  of  the 
soil  is  a  powerful  factor  in  determining  the  amount  of  ammonia 
produced  in  soils  by  pure  cultures  of  organisms  possessed  of 
ammonifying  powers,  if  the  fifteen  different  organisms  used  are 
a  suitable  criterion.  This  confirms  the  findings  of  J.  G.  Lipman 
in  his  long  series  of  ammonification  experiments  with  mixed  cul- 
tures. For  practical  purposes,  we  may  add  that  most  of  the 
bacteria  ammonify  dried-blood  nitrogen  equally  well  in  the  clay- 
adobe  soil  and  in  clay-loam  soil,  though  there  does  appear  to  be 
a  slight  though  consistently  greater  amount  of  ammonia  produced 
in  the  first-named  soil.  Again,  we  find  in  the  clay-adobe  soil 
an  organism  which  stands  out  as  far  superior  to  all  others  in 
ammonifying  efficiency  and  again  also  it  is  not  the  same  organism 
as  manifested  that  superiority  in  the  preceding  soil.  While  the 
duplicate  determinations  here  do  not  agree  as  well  as  might  be 
desired,  they  indicate  amounts  so  much  greater  than  the  quan- 
tities of  ammonia  produced  by  the  other  organisms  of  the  series 
that  there  can  be  no  doubt  of  the  marked  and  superior  efficiency 
of  B.  tumescens  as  an  ammonia  producer  from  the  nitrogen  of 
dried  blood  in  the  clay  adobe  soil  as  a  medium.  Two  other 
organisms  appear  to  be  in  the  second  class  in  this  series  and  they 
are  Mic.  tetragenus  and  B.  mycoides.  The  first,  it  can  be  seen 
from  Table  III,  occupied  third  place  in  efficiency  in  the  clay 
loam  soil,  but  the  second  has  thus  far  been  relatively  inefficient. 
All  the  other  organisms  of  the  series  do  not  manifest  differences 
in  efficiency  of  sufficient  magnitude  to  warrant  further  comment, 
except  that  it  is  curious  and  interesting  to  note  that  the  most 
efficient  organism  in  the  sandy  soil  is  the  least  efficient  organism 
in  the  adobe  soil. 

It  should  be  remarked  here  that  the  generally  excellent  agree- 
ment between  duplicate  determinations,  as  shown  in  the  table. 


148        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

eliminates  the   fear  that  physical  conditions  in  the   individual 
cultures  might  operate  to  produce  the  rather  marked  effects  noted. 

In  a  comparison  of  the  three  soils  it  appears  that  the  follow- 
ing organisms  are  among  those  which  show  the  highest  efficiency 
in  transforming  the  nitrogen  of  dried  blood  into  ammonia:  Ps. 
putida,  Sarcina  lutea,  B.  vulgaris,  B.  proteus  vulgaris,  Mic. 
tetragenus,  B.  tumescens.  Of  these  organisms  only  Mic.  t et ra- 
ge nus  shows  a  high  efficiency  in  all  three  soils.  Of  the  others 
B.  vulgaris  shows  a  high  efficiency  in  both  the  sandy  and  adobe 
soils  and  Ps.  putida  in  the  sandy  and  clay-loam  soil.  The  rest 
namely  Sarcina  lutea,  B.  proteus  vulgaris  and  B.  tumescens,  are 
markedly  efficient  only  in  one  soil  each,  namely,  the  sandy  soil  for 
the  first,  the  clay-loam  soil  for  the  second,  and  the  clay-adobe 
soil  for  the  third.  It  appears  to  us  particularly  worthy  of  note, 
also,  that  of  the  last  three  organisms  named  B.  proteus  vulgaris 
and  B.  tumescens  each  holds  a  pre-eminent  position  of  efficiency 
in  its  own  soil  which  no  other  organism  of  the  fifteen  has 
approached;  and  even  in  the  case  of  Sarcina  lutea  we  find  that 
it  occupies  a  position  in  efficiency  in  its  favorite  soil  which  is 
second  only  to  that  of  B.  vulgaris  and  not  very  far  behind  the 
latter.  This  rather  remarkable  condition  would  hardly  seem  to 
be  accidental  and  appears  to  us  to  indicate  for  certain  organisms 
marked  preferences  for  certain  physical  characteristics  in  media 
in  which  they  are  grown. 

It  must  be  added  here,  too,  that  only  six  of  the  fifteen 
organisms  show  marked  ammonifying  efficiency  as  regards  the 
nitrogen  of  dried  blood  even  when  tested  in  three  widely  different 
soil  types.  The  others  vary  but  little  from  one  another  in  all 
soils.  Oddly  enough,  the  organism  which  has  shown  the  highest 
ammonifying  efficiency  because  it  maintained  it  through  all  soils, 
namely,  Mic.  tetragenus,  has  never  before  been  looked  upon,  so 
far  as  we  are  aware,  as  an  important  ammonifier.  That  it  should 
be  the  only  one  of  fifteen  organisms  tested  which  should  be  about 
equally  efficient  in  all  soils  used  is  not  unworthy  of  note. 

Tt  is  further  significant  to  note  here  that  B.  mycoides,  which 
has  always  been  regarded  as  one  of  the  most  efficient  soil  organ- 
isms at  ammonification,  does  not  in  our  experiments  show  any 
unusual  activity  in  that  direction,  at  any  rate  when  dried  blood 


1914]        Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures  149 

is  used  as  the  ammonifiable  material,  no  matter  what  the  soil 
medium.  A  possible  exception  to  this  statement  may  be  found 
in  the  case  of  the  adobe  soil,  in  which  B.  mycoides  seems  to  be 
superior  to  all  but  three  or  four  of  the  fifteen  organisms  tested. 
Such  superiority,  under  the  circumstances  noted,  is  probably  of 
little  significance. 


Series  II.  Experiments  with  Tankage 
This  series  was  arranged  in  a  manner  similar  to  the  preced- 
ing, the  sandy,  clay  loam,  and  clay-adobe  soils  again  being  used 
as  media,  but  the  ammonifiable  material  in  this  case  was  a  high- 
grade  tankage,  the  nitrogen  content  of  which  was  9.62  per  cent. 
The  results  of  the  ammonia  determinations  were  as  shown  in 
Table  IV  on  page  150. 

Discussion  of  Series  II 

The  Sandy  Soil 

When  we  study  in  the  foregoing  table,  the  ammonification  of 
tankage  by  pure  cultures  of  bacteria,  and  compare  the  results 
with  those  of  Table  III,  we  see  at  once  some  very  striking  differ- 
ences between  the  ability  of  the  same  micro-organisms  to  produce 
ammonia  from  tankage  and  from  dried  blood  respectively.  Not 
only  do  more  of  the  organisms  show  a  high  efficiency  in  trans- 
forming the  tankage  nitrogen  to  ammonia,  but  the  point  of 
highest  efficiency  is  not  reached  by  the  same  organisms  as  before, 
others  having  taken  their  places  in  this  series  with  sandy  soil  as 
the  culture  medium.  For  example,  we  find  that  B.  mesenteric  us, 
which  in  the  preceding  series  showed  throughout  an  extremely 
low  ammonifying  efficiency  even  in  the  sandy  soil,  now  manifests 
in  the  same  culture  medium,  which,  however,  has  tankage  in 
place  of  blood  added  to  it,  the  highest  efficiency  of  all  of  the 
organisms  tested.  Indeed,  it  occupies  a  position  of  its  own  in 
that  direction,  much  as  does  B.  vulgaris  in  the  sandy  soil  of  the 
last  series.  The  organism  which  approaches  it  most  closely  in 
the  same  medium  is  B.  proteus  vulgaris  which,  however,  falls  25 
per  cent  short  of  producing  the  amount  of  ammonia  yielded  by 


150        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 


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1914]        Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures  151 

B.  mesentericus.  Then  follow,  not  far  behind  B.  proteus  vulgaris, 
B.  tumescens,  B.  ramosus,  and  B.  vulgaris  in  the  order  named, 
the  latter  being  more  than  33%  per  cent  short  of  the  efficiency 
exhibited  by  B.  mesentericus.  Nearly  all  of  the  other  organisms 
fall  more  than  50  per  cent  short  of  the  efficiency  of  the  last- 
named  organism  under  this  set  of  circumstances,  namely,  sandy 
soil  with  tankage.  There  are,  therefore,  but  five  organisms  out  of 
the  fifteen  tested  which  can  be  adjudged  distinctly  efficient 
ammonifying  organisms  under  these  conditions.  B.  mycoides 
again  exhibits  a  low  efficiency,  and  not  far  different  from,  though 
slightly  below,  that  manifested  by  it  in  the  same  soil  in  the  dried- 
blood  series. 

It  must  be  added  here  that,  with  the  exception  of  B.  mesen- 
tericus, the  efficient  organisms  in  this  part  of  Series  II  have  also 
shown  more  or  less  marked  efficiency  in  the  preceding  series. 


The  Clay-Loam  Soil 

Most  of  the  organisms  tested  in  the  clay-loam  soil  seem  to 
have  found  the  latter  a  more  congenial  medium  for  ammonifica- 
tion with  tankage  than  they  did  when  dried  blood  was  present. 
In  this  part  of  Series  II,  three  of  the  organisms,  namely,  B. 
mesentericus,  B.  vulgatus,  and  B.  tumescens,  were  not  only  the 
most  efficient  ammonifiers  but  also  about  equal  in  their  ammoni- 
fying power.  It  appears,  therefore,  that  so  far  as  tankage  is 
concerned  B.  mesentericus  is  an  equally  efficient  ammonifier  in 
the  clay-loam  and  sandy  soils.  B.  tumescens  has  previously 
established  its  pre-eminent  position  among  the  fifteen  organisms 
as  an  ammonifier  of  dried-blood  nitrogen  in  the  adobe  soil,  but 
B.  vulgatus  enters  here  for  the  first  time  as  a  markedly  efficient 
ammonifier. 

Only  slightly  behind  the  three  organisms  just  discussed  in 
their  ammonifying  efficiency  as  regards  tankage  nitrogen  in  the 
clay-loam  soil  are,  in  the  order  named,  B.  subtilis,  B.  mycoides, 
and  B.  vulgaris.  While  the  latter  has  manifested  its  high 
efficiency  in  other  series  above  described,  the  first  two  organisms 
named  for  the  first  time  in  the  work  thus  far  described  show 
marked  ammonifying  ability.    Only  three  organisms  in  this  part 


152        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

of  Series  II  have  shown  themselves  to  be  really  weak  ammonifiers, 
and  they  are  B.  proteus  vulgaris,  B.  icteroides,  and  Mic.  tetra- 
genus.  The  first  and  the  third  of  these,  it  will  be  remembered, 
have  given  evidence  of  marked  efficiency  under  other  circum- 
stances, but  the  second  has  thus  far  been  throughout  an  organism 
of  low  efficiency.  It  is  striking  to  note  the  much  greater  uni- 
formity which  exists  in  this  portion  of  Series  II  in  the  ammoni- 
fying powers  of  four-fifths  of  the  organisms  tested  than  that 
which  obtains  in  other  parts  of  this  series  and  of  other  series. 


The  Clay-Adobe  Soil 

Here  again  we  find  the  great  uniformity  in  ammonifying 
power  between  the  larger  number  of  bacteria  tested  which  is 
characteristic  of  the  foregoing  section  of  Series  II.  On  the  other 
hand,  the  clay-loam  soil  seems  to  have  been  a  more  congenial 
medium  than  the  adobe  soil  for  the  ammonification  of  tankage 
nitrogen,  for  larger  amounts  of  ammonia  are  produced  in  it  by 
the  same  organisms  in  the  same  period  of  incubation. 

Sarcina  lutea  shows  the  highest  efficiency  as  an  ammonifier 
in  this  part  of  the  series,  but  is  only  slightly  more  efficient  than 
B.  tumescens.  The  organism  taking  third  place  is  B.  mycoides 
which,  indeed,  is  not  far  behind  the  other  two.  Next  in  order  of 
importance  and  still  very  efficient  ammonifiers  are  B.  subtilis  and 
Streptothrix,  sp.  The  other  organisms  are  considerably  weaker 
ammonifiers  than  those  just  mentioned  and  B.  icteroides  again 
proves  to  be  distinctly  the  weakest.  Both  Sarcina  lutea  and 
B.  tumescens  have,  as  mentioned  above  under  other  circum- 
stances, plainly  evidenced  their  high  efficiency  as  ammonifiers 
and  B.  mycoides  and  B.  subtilis  have  taken  similar  positions  with 
respect  to  tankage  in  the  clay-loam  soil.  Streptothrix,  sp.,  it  will 
be  remembered,  has  also  shown  a  high  ammonifying  power  before 
in  the  case  of  the  clay-loam  soil  when  dried  blood  was  used. 

Comparing  the  three  soils  in  this  series  with  the  same  ones 
in  the  preceding  series,  it  appears  quite  clear  that  taking  them 
by  and  Large,  ammonifying  bacteria  manifest  a  much  higher 
efficiency  with  high-grade  tankage  than  with  dried  blood  under 
similar  conditions.    Likewise  also,  in  Series  II  a  small  number  of 


1914]        Lipman-Burgess:  Ammonification  in  Soils  by  Pure  Cultures      ,      153 

organisms  mostly  identical  with  those  in  Series  I  seem  to  mani- 
fest a  distinctly  high  efficiency  which  the  much  larger  number 
of  the  balance  do  not  in  most  cases  even  approach.  It  is  worthy 
of  remark,  moreover,  that  B.  mycoides  attains  or  rather  ap- 
proaches in  portions  of  Series  II  its  position  of  prime  importance 
which  has  thus  far  so  generally  been  accorded  it  among  ammoni- 
fying organisms. 

Series  III.    Cottonseed  Meal 

The  cottonseed  meal  used  in  this  series  showed  on  analysis  a 
nitrogen  content  equal  to  5.5  per  cent.  The  experiment  in  this 
series  was  otherwise  conducted  like  those  of  Series  I  and  II, 
2  per  cent  of  cottonseed  meal  being  used,  or  1  gram  per  50  grams 
of  soil.    The  results  are  shown  in  Table  III. 

Again  the  efficiency  of  the  organisms  tested  is  much  greater 
in  the  sandy  soil  than  in  either  the  clay-loam  or  adobe  soils. 

Sandy  Soil 

In  this  part  of  Series  III  the  organism  of  greatest  ammonify- 
ing efficiency  is  B.  tumescens.  Those  approaching  it  closely  in 
efficiency  are  B.  vulgatus,  Sarcina  luiea,  and  B.  mycoides  in  the 
order  named.  B.  ramosus  takes  fifth  place  and  the  next  three 
organisms  fall  more  than  33 %  per  cent  short  of  producing  the 
amount  of  ammonia  yielded  by  B.  tumescens.  They  are  B. 
mesentericus,  B.  megatherium,  and  B.  proteus  vulgaris.  The 
balance  of  the  organisms  show  only  about  half  the  efficiency  of 
B.  tumescens  and  in  one  case,  B.  icteroides,  which  has  in  all  pre- 
vious series  shown  a  very  low  efficiency,  only  about  25  per  cent 
of  the  maximum  efficiency  is  manifested.  Taken  as  a  whole,  the 
data  given  in  Table  III  for  the  Anaheim  sandy  soil  reflects 
favorably  on  cottonseed  meal  as  a  source  of  available  nitrogen 
under  the  conditions  named.  Again,  the  greatest  efficiency  is 
manifested  in  this  section  of  Series  III  by  organisms  which  in  all 
cases  have  shown  high  efficiency  in  other  series  above  reported. 
As  in  the  case  of  the  heavy  soil  in  the  preceding  series  with 
tankage,  B.  mycoides  also  shows  marked  efficiency  in  the  light  soil 
when  cottonseed  meal  is  used. 


154        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  i 


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1914]       Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures  155 

The  Clay-Loam  Soil 
The  most  notable  thing  in  this  part  of  Series  III  is  as  above 
intimated,  the  very  low  efficiency  of  all  of  the  organisms  tested. 
Indeed  only  two  organisms  manifest  any  notable  activity  as 
ammonifiers  of  nitrogen  in  cottonseed  meal  in  the  clay-loam  soil 
as  a  medium.  These  two  in  the  order  of  their  importance  are 
B.  ramosus  and  Streptothrix,  sp.  To  these,  in  view  of  the  dis- 
agreement of  the  duplicates  as  above  shown,  B.  vulgaris  may 
probably  be  added,  and  perhaps  also  B.  vulgatus.  The  other 
organisms  are  all  distinctly  below  the  first  two  mentioned,  and 
the  lowest  efficiency  thus  far  noted  is  that  exhibited  by  Mic. 
tetragenus  in  this  soil.  Considering  the  high  efficiency  of  the 
latter  organism  in  Series  I,  the  results  just  discussed  are  puzzling. 

The  Clay-Adobe  Soil 

Even  a  casual  glance  at  the  data  obtained  in  the  clay-adobe 
soil  as  a  medium  indicates  the  distinct  superiority  of  that  medium 
to  the  clay  loam  soil  for  ammonification  of  the  nitrogen  of  cotton- 
seed meal.  Not  only  relatively  but  absolutely  the  data  obtained 
show  the  production  of  much  larger  amounts  of  ammonia  in  this 
portion  of  the  work. 

As  is  the  case  in  the  sandy  soil  with  cottonseed  meal  as  the 
ammonifiable  material,  B.  tumescens  shows  its  distinct  superiority 
to  the  other  organisms  as  an  ammonifier  in  the  adobe  soil.  The 
next  most  efficient  organism  is  B.  subtilis,  which,  however,  is  con- 
siderably less  efficient ;  and  the  next  two  organisms,  about  as  far 
below  B.  subtilis  in  efficiency  as  the  latter  is  below  B.  tumescens, 
are  B.  ramosus  and  B.  mycoides.  The  other  organisms  are  all 
low  in  efficiency,  though  in  nearly  all  cases  absolutely  better  than 
the  same  organisms  in  the  clay-loam  soil.  Thus  far  we  find  that 
B.  subtilis  shows  itself  markedly  efficient,  for  the  first  time,  in 
the  clay-adobe  soil  with  cottonseed  meal.  All  the  other  organisms 
above  named  have  manifested  marked  efficiency  in  some  parts 
of  the  foregoing  series. 

Looking  at  Series  III  as  a  whole  it  is  interesting  to  note  that 
the  nitrogen  of  cottonseed  meal  seems  to  be  made  available 
through  the  activity  of  pure  cultures  of  ammonifying  bacteria, 


156        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

with  much  greater  rapidity  than  has  heretofore  been  believed. 
The  next  striking  fact  brought  out  in  these  results  is  the  clearly 
indicated  superiority  of  the  clay-adobe  soil  to  the  clay-loam  soil 
as  a  medium  for  the  ammonification  of  the  nitrogen  in  cottonseed 
meal.  The  third  point  worthy  of  mention  in  Series  III  is  the  fact 
that  we  find  again  in  it,  as  in  the  preceding  series,  only  a  few 
of  the  fifteen  organisms  tested  which  show  marked  ammonifying 
efficiency. 

The  culture  of  Ps.  fluorescens  died  in  the  midst  of  these 
investigations  and  was  not  replaced. 


Series  IV.    Fish  Guano 

Fish  guano,  in  accordance  with  the  teachings  of  Voorhees  and 
other  agricultural  chemists,  has  always  been  esteemed  a  good 
source  of  available  nitrogen;  in  the  words  of  Voorhees,2  "rank- 
ing in  availability  well  up  to  blood  and  tankage."  It  seemed  to 
us  therefore  of  importance  to  compare  in  these  pure  culture 
studies  fish  guano  with  the  other  organic  materials  discussed 
above.  Accordingly  a  series  was  started  similar  to  those  above 
described,  except  that  1%  grams  of  finely  sifted  fish  guano  was 
the  ammonifiable  material  used  per  50  grams  of  soil.  The  fish 
guano  used  contained  8.63  per  cent  nitrogen.  The  results 
obtained  are  shown  in  Table  VI. 

The  data  in  Table  VI  not  only  seem  to  confirm  the  opinion  of 
Voorhees  as  above  stated,  if  ammonia  production  by  pure  cul- 
tures may  be  taken  as  a  criterion  for  determining  the  availability 
of  fish  guano,  but  they  indicate  in  most  striking  fashion  what 
was  not  shown  in  any  of  the  foregoing  series  for  the  fertilizers, 
and  particularly  in  the  sandy  soil,  namely,  the  obliteration  of 
the  marked  physiological  differences  obtaining  in  other  series 
between  the  different  organisms.  Of  the  four  nitrogenous  fer- 
tilizers thus  far  discussed,  fish  guano  seems  to  contain  the  form 
of  nitrogen  most  generally  ammonified  by  a  large  group  of 
bacteria. 


1914]        Lipman-Burgess :  Ammoniftcation  in  Soils  by  Pure  Cultures  157 


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]  58        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

The  Sandy  Soil 
The  good  ammonifying  power  manifested  by  most  of  the 
organisms  tested  in  this  soil  with  fish  guano  finds  its  maximum 
in  the  case  of  B.  vulgaris,  which,  it  will  be  remembered,  has 
already  given  evidence  of  notable  ammonifying  efficiency  with 
other  nitrogenous  fertilizers.  B.  tumescens,  likewise  an  organism 
with  a  record  as  an  ammonifier  well  established,  is  a  very  close 
second  to  B.  vulgaris  in  its  efficiency  at  the  ammonification  of  the 
nitrogen  in  fish  guano.  Four  other  organisms  distinctly  in  the 
second  class,  and  about  alike  in  ammonifying  efficiency  in  this 
part  of  Series  IV,  are,  in  the  order  of  their  efficiency,  Ps.  putida, 
B.  vulgatus,  B.  ramosus,  and  Sarcina  lutea.  Peculiarly  enough, 
B.  icteroides  shows  an  ammonifying  efficiency  here  which  places 
it  in  the  second  class,  a  position  Which  it  has  thus  far  never  even 
remotely  approached  in  the  series  of  experiments  above  discussed, 
nor,  for  that  matter,  in  other  series  which  are  described  below. 
All  other  organisms  in  this  part  of  Series  IV  are  distinctly  in 
the  third  class,  but  nevertheles  manifest  notable  ammonifying 
efficiency. 

In  no  other  series  of  results  have  we  obtained  such  sharp 
lines  of  demarcation  between  the  classes  of  organisms  here  tested, 
and  arbitrarily  grouped,  in  accordance  with  their  respective 
powers  of  transforming  organic  nitrogen  into  ammonia. 

The  Clay -Loam  Soil 

The  change  in  the  physical  condition  of  the  soil  from  the  sand 
to  the  clay  loam  shows  a  marked  effect  on  the  ammonifying 
power  of  the  same  organisms.  Nevertheless  we  find,  on  the  whole, 
the  best  set  of  results  thus  far  obtained  with  the  clay  loam  when 
the  series  with  fish  guano  is  studied.  In  a  class  by  itself  under 
these  conditions  is  B.  megatherium,  which  is  markedly  superior 
to  all  other  organisms  in  this  part  of  Series  IV,  except  B.  mesen- 
tericus,  which  is  a  close  second.  The  organism,  however,  which 
takes  third  place,  B.  vulgaris,  falls  about  20  per  cent  short  of 
m11  fining  the  efficiency  of  B.  megatherium  under  these  conditions. 
Distinctly  lower  in  efficiency  in  this  part  of  Series  IV  are,  in 
the  order  of  their  importance,  but  only  slightly  different  from 


1914]        Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures  159 

one  another,  B.  vulgatus,  Ps.  putida,  B.  ramosus,  and  B.  tumes- 
cens.  All  of  these,  however,  fall  more  than  30  per  cent  short  of 
attaining  the  ammonifying  efficiency  of  B.  megatherium  under 
the  conditions  here  considered.  Two  other  organisms,  B.  subtilis 
and  Sarcina  lutea,  fall  40  per  cent  short  of  the  efficiency  of  B. 
megatherium,  and  the  others  all  fall  far  below  even  that  figure. 
It  is  singular,  here  again,  .that  the  physical  nature  of  the  soil 
medium  employed  should  so  strikingly  and  so  variously  influence 
the  efficiency  of  the  ammonifying  bacteria.  The  two  organisms 
which  are  distinctly  superior  in  ammonifying  ability  as  regards 
the  nitrogen  of  fish  guano  in  the  clay  loam  soil  were  only  of 
moderate  efficiency  with  the  same  form  of  nitrogen  in  the  sandy 
soil.  And  again,  the  organism  which  in  the  latter  soil  was 
paramount  in  its  position  retreats  in  the  clay-loam  soil  to  third 
place. 

The  Clay-Adobe  Soil 

This  is  the  first  series  of  those  we  have  thus  far  considered, 
as  the  data  in  Table  VI  shows,  in  which  the  clay-adobe  soil  proves, 
on  the  whole,  to  be  inferior  to  the  clay-loam  soil.  Seven  organ- 
isms out  of  fourteen  here  show  an  extremely  low  ammonifying 
efficiency,  and  two  others  are  by  no  means  efficient  organisms. 
That  leaves  five  organisms  in  this  group  which  may  be  considered 
of  importance.  Of  these,  B.  vulgaris  is  the  most  efficient,  but 
B.  tumescens  is  not  far  behind  it.  B.  subtilis  belongs  to  the 
second  class  in  this  group  and  B.  proteus  vulgaris  and  B. 
mycoides  to  the  third  class.  It  is  rather  unfortunate  that  it  was 
not  possible  in  this  part  of  Series  IV  to  obtain  better  agreement 
between  duplicate  determinations.  It  would  not  appear  to  us, 
however,  that  the  discrepancies  in  question  militate  against  the 
justice  of  the  conclusions  above  drawn. 

B.  megatherium  holds  a  very  good  place  in  the  sandy  and 
clay  loam  soils  of  Series  IV  and  a  fair  place  in  the  adobe  soil. 
The  same  is  even  more  strikingly  true  of  B.  vulgaris,  and  in  a 
minor  degree  this  is  also  true  of  B.  tumescens.  The  other  organ- 
isms do  not  manifest  such  consistent  efficiency  under  the  three 
widely  varying  soil  conditions. 


160        University  of  California  Publications  in  Agricultural  Sciences     [Vol.  1 

Miscellaneous  Series 

For  the  purpose  of  comparing  sheep  and  goat  manure  as  well 
as  phosphatie  guano  with  the  other  organic  materials  above 
described  and  with  peptone,  it  was  deemed  of  interest  to  obtain 
data  exemplifying  the  ammonification  in  the  same  soil  of  all  the 
different  materials  above  used  with  the  three  additional  ones  just 
mentioned.  The  sandy  soil  was  chosen  for  this  series  and  when 
peptone  (Witte)  was  used,  0.5  gram  of  it  was  added  to  50  grams 
of  soil.  The  peptone  contained  14.14  per  cent  N.  The  sheep 
and  goat  manure  (2.13  per  cent  N),  owing  to  its  low  nitrogen 
content,  was  added  to  the  extent  of  3  grams  per  50  grams  of  soil, 
and  the  phosphatie  guano  (3.96  per  cent  N)  was  also  added  at 
the  rate  of  3  grams  per  50  grams  of  soil.  The  results  obtained 
are  recorded  in  Table  VII. 

As  was  to  be  expected,  the  very  available  form  of  nitrogen 
in  the  peptone  allows  of  the  production  of  much  larger  quantities 
of  ammonia  than  do  the  less  available  forms  of  the  other  materials. 
The  fineness  of  division  and  easy  solubility  of  the  peptone,  as  well 
as  the  form  of  nitrogen  which  it  contains,  doubtless  have  con- 
tributed to  the  results.  The  more  or  less  uniform  decomposition 
of  it,  however,  by  most  of  the  organisms  tested  shows  peptone  to  be 
unsuited,  as  has  been  claimed  by  other  investigators,  for  ammoni- 
fication studies  with  pure  or  mixed  cultures  when  the  application 
of  the  data  obtained,  to  field  conditions,  is  contemplated.  How- 
ever that  may  be,  Sarcina  lutea  shows  the  highest  efficiency  at 
ammonifying  peptone  nitrogen  and  at  least  six  other  organisms 
approach  it  rather  closely.  Relatively  speaking,  all  but  two  of 
the  organisms  tested  are  efficient  ammonifiers  of  peptone  nitrogen. 
But  their  position  with  respect  to  peptone,  as  can  be  seen  from 
Table  VII,  is  no  criterion  as  to  their  efficiency  with  respect  to 
the  other  materials. 

One  of  the  interesting  facts  about  the  ammonification  of  the 
sheep  and  goat  manure  by  pure  cultures  is  that  only  one  organism 
showed  ammonifying  efficiency  worthy  of  the  name,  and  that  was 
B.  megatherium.  The  other  organisms  showed  a  very  slight 
power  only  of  ammonifying  the  nitrogen  in  it.  Just  why  this 
large  discrepancy  should  exist  with  respect  to  this  manure 
between  B.  megatherium  and  the  other  organisms  still  remains 


1914]       Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures 


161 


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162        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

to  be  explained.  If,  therefore,  the  readiness  of  the  transformation 
of  its  nitrogen  into  ammonia  by  pure  cultures  of  ammonifiers  is 
to  be  taken  as  a  criterion,  sheep  and-  goat  manure  must  be 
adjudged  to  contain  a  relatively  unavailable  form  of  nitrogen. 
The  most  amazing  evidence  portrayed  in  Table  VII  is  the 
very  high  availability  of  the  nitrogen  of  bat  guano,  which  is  a 
phosphatic  guano.  "While  without  question  the  fact  of  the  large 
amount  of  the  guano  used,  as  well  as  its  low  content  of  nitrogen, 
preclude  an  accurate  and  wholly  justifiable  comparison  of  it  with 
the  other  nitrogenous  materials,  one  cannot  help  being  struck 
by  the  large  transformation  of  its  nitrogen  into  ammonia  which 
nearly  all  of  the  organisms  tested  can  accomplish.  In  many 
ways,  the  transformation  of  the  nitrogen  of  bat  guano  into 
ammonia  resembles  that  of  the  transformation  of  peptone 
nitrogen.  The  differences  between  the  ammonifying  powers  of 
the  different  organisms  are,  however,  unquestionably  more 
marked  in  the  case  of  the  bat  guano.  In  its  efficiency  as  an 
ammonifier  of  bat  guano  nitrogen,  B.  mycoides  appears  for  the 
first  time  in  all  the  series  studied  to  be  distinctly  superior  to  all 
the  other  organisms  tested.  B.  vulgaris  easily  takes  second  place. 
Not  far  behind,  however,  and  about  equal  in  efficiency,  are  B. 
megatherium,  B.  vulgatus,  B.  tumescens,  and  Mic.  tetragenus. 
In  the  third  class  are  B.  ramosus,  B.  subtilis,  and  Ps.  putida,  B. 
proteus  vulgaris,  and  B.  mesentericus.  In  the  fourth  class  are 
Streptothrix,  sp.,  Sarcina  lutea,  B.  icteroides,  and  Ps.  fluorescens. 


A  Comparison  of  the  Relative  Availabilities  of  the  Organic 

Materials  above  Employed  based  on  the  Percentage 

of  Nitrogen  Contained  in  Them  that  was 

Transformed  to  Ammonia 

Thus  far  we  have  been  considering  only  the  relative  degrees  of 
efficiency  as  ammonifiers  of  the  different  organisms  among  them- 
selves as  respecting  a  given  organic  form  of  nitrogen  in  a  given 
soil.  There  is  possible,  however,  a  further  very  interesting  study 
of  the  data  above  given  as  a  basis.  We  refer  to  the  percentage 
of  nitrogen  which  is  transformed  in  the  different  materials  into 
ammonia  so  as  to  give  them  a  relative  rating  as  to  availability  as 


1914]       Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures 


163 


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164        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

regards  the  work  of  pure  cultures.  The  columns  on  availability 
of  the  various  tables  show  a  comparison  in  different  soils  of  all 
the  materials  as  attacked  by  the  same  organisms,  and  give  the 
percentages  of  nitrogen  in  those  materials  which  were  trans- 
formed to  ammonia.  In  Table  VIII  all  of  the  columns  indicating 
percentages  of  nitrogen  of  different  fertilizers  made  available 
are  brought  together  from  the  other  tables  and  one  is  enabled  to 
compare  with  much  greater  ease  the  different  materials  on  the 
basis  of  availability  in  the  same  soil  and  with  the  same  organism 
and  in  different  soils,  with  different  organisms. 

The  Sandy  Soil 

So  far  as  this  soil  is  concerned,  the  data  in  Table  VIII 
indicate  clearly  the  superiority,  from  the  point  of  view  of  the 
availability  of  its  nitrogen,  of  cottonseed  meal  to  the  other  organic 
nitrogenous  fertilizers  with  which  it  is  compared.  Not  only  are 
the  absolute  amounts  of  ammonia  produced  in  most  cases  larger 
from  cottonseed  meal  nitrogen  than  from  other  forms,  but  there 
are  more  organisms  of  the  fifteen  tested  which  can  vigorously 
ammonify  this  form  of  nitrogen.  So  that  to  illustrate,  there  are 
but  five  organisms  which  have  shown  the  power  to  transform 
15  per  cent  or  more  of  the  nitrogen  of  dried  blood  into  ammonia 
in  the  sandy  soil.  Under  similar  conditions  there  are  ten 
organisms  which  hold  such  a  record  for  cottonseed  meal.  Tank- 
age shows  itself  to  be  the  next  important  nitrogenous  fertilizer 
to  cottonseed  meal  from  the  point  of  view  of  the  availability  of 
its  nitrogen.  Thus,  comparing  it  with  dried  blood  as  above,  we 
find  that  there  are  nine  organisms  which  transform  15  per  cent 
or  more  of  the  nitrogen  of  tankage  into  ammonia.  No  such  high 
availability  is  obtained  at  all  in  the  case  of  the  fish  guano. 

When  we  consider  these  nitrogenous  fertilizers  from  the  point 
of  view  of  the  transformation  of  10  per  cent  or  more  of  their 
nitrogen  into  ammonia,  we  find  that  there  are  thirteen  of  the 
fifteen  organisms  tested  which  possess  that  power  as  regards 
cottonseed  meal  nitrogen  and  another  comes  very  close  to  that 
point.  In  the  case  of  tankage  nitrogen  there  are  thirteen  organ- 
isms with  a  similar  power.     In  the  case  of  dried-blood  nitrogen 


]  914]        Lipman-Burgess :  Ammonification  in  Soils  by  Pure  Cultures  165 

there  are  but  ten  organisms  which  can  accomplish  that  task,  and 
but  seven  such  in  the  case  of  fish-guano  nitrogen. 

From  the  point  of  view  of  availability  by  pure  cultures 
therefore  in  the  sandy  soil  the  four  nitrogenous  fertilizers  are 
to  be  rated  as  follows :  cottonseed  meal,  tankage,  dried  blood,  and 
fish  guano.  The  first  two  are  nearly  alike  and  are  far  superior 
to  the  last  two,  which  are  nearly  alike,  but  much  more  different 
from  each  other  than  the  first  two. 

The  greatest  efficiency  at  ammonification  manifested  by  any 
organism  in  the  sand}^  soil  is  that  of  B.  mesentericus  with  tankage 
nitrogen,  which  transforms  32.52  per  cent  of  the  nitrogen  present 
into  ammonia  in  twelve  days.  It  should  be  noted  in  this  con- 
nection also  that  absolutely  higher  amounts  of  ammonia  are 
produced  from  tankage  nitrogen  than  from  any  other  form  in 
the  sandy  soil,  even  if  there  are  fewer  organisms  which  attack 
it  readily  than  there  are  in  the  case  of  the  cottonseed  meal. 


The  Clay -Loam  Soil 

Conditions  are  evidently  entirely  different  for  ammonification 
in  this  soil.  Not  only  is  the  ammonia  production  very  low  so 
far  as  all  the  fertilizers  are  concerned,  but  they  no  longer  bear 
to  one  another  the  relation  which  obtained  between  them  in  the 
sandy  soil.  There  are  thus  but  few  organisms  which  possess  the 
power  of  transforming  10  per  cent  or  more  of  the  total  nitrogen 
in  any  of  the  four  fertilizers  into  ammonia  in  twelve  days.  In 
fact,  there  are  none  such  in  the  case  of  the  dried  blood,  only  two 
such  each  in  the  cases  of  cottonseed  meal  and  fish  guano,  and 
seven  such  in  the  case  of  the  tankage.  No  organism  attains  to 
the  production  of  ammonia  equivalent  to  13  per  cent  of  the  total 
amount  present  in  the  clay-loam  soil  regardless  of  the  kind  of 
fertilizer  at  its  disposal. 

The  tankage,  however,  is  superior  to  the  cottonseed  meal  in 
the  clay  loam  and  distinctly  so  as  above  indicated.  The  cotton- 
seed meal  takes  second  place,  the  fish  guano  third  place,  and  the 
dried  blood  is  by  far  the  poorest.  Indeed,  no  organism  was 
capable  of  producing  an  amount  of  ammonia  in  excess  of  6.91 


166        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

per  cent  of  the  total  amount  of  dried-blood  nitrogen  furnished, 
and  that  occurred  in  only  one  case,  all  the  other  organisms  pro- 
ducing much  less. 

The  Clay-Adobe  Soil 

Some  very  striking  facts  become  apparent  when  the  avail- 
ability of  the  four  fertilizers  in  clay-adobe  soil  are  considered. 
While  on  the  whole  dried-blood  nitrogen  is  only  slightly  more 
efficiently  transformed  into  ammonia  than  in  the  clay-loam  soil, 
cottonseed  meal  and  tankage,  particularly  the  former,  are  more 
vigorously  acted  on  in  the  clay-adobe  soil  by  most  of  the  organ- 
isms. Fish  guano,  while  not  markedly  so,  is  none  the  less  superior 
here  again  to  dried  blood. 

Again  comparing  the  different  fertilizers  on  the  basis  of  the 
amounts  of  their  nitrogen  transformed  into  ammonia  by  the 
organisms  tested,  we  find  that  four  organisms  transform  10  per 
cent  or  more  of  the  nitrogen  in  cottonseed  meal  into  ammonia, 
a  like  number  accomplish  similar  results  in  the  case  of  tankage, 
and  none  succeeds  in  that  direction  in  either  fish  guano  or  dried 
blood.  While  thus  cottonseed  meal  and  tankage  appear  alike, 
a  study  of  table  VIII  reveals  the  superiority  of  the  former  in  the 
larger  absolute  amounts  of  nitrogen  which  are  transformed  there 
than  in  the  case  of  the  latter. 

The  first  striking  fact  shown  in  Table  VIII  is  the  marked 
superiority  of  the  sandy  soil  as  a  medium  for  ammonification  by 
pure  cultures.  Likewise  the  added  fact  of  its  superiority  as  a 
medium  for  most  of  the  organisms  tested  must  be  noted  in  this 
connection.  The  second  point  of  great  interest  is  the  surprising 
fact  of  the  superiority  of  the  clay-adobe  soil  to  the  clay-loam 
soil  as  a  medium  for  ammonification.  From  its  tenacious  nature 
one  would  suppose  the  former  type  to  be  a  much  poorer  medium 
for  ammonification  than  the  clay  loam  and  yet  it  is  distinctly 
superior  to  the  latter  as  regards  tankage  and  cottonseed  meal. 
As  regards  fish  guano,  it  is  slightly  inferior  to  the  clay  loam  and 
again  as  regards  the  dried  blood  about  equal  to  the  clay  loam 
or  possibly  slightly  superior. 

Considering  all  the  data  given  in  Table  VIII  from  all  points 
of  view,  tankage  must  be  given  first  place  as  regards  the  avail- 


1914]       Lipman- Burgess :  Ammonification  in  Soils  by  Pure  Cultures  167 

ability  of  its  nitrogen,  cottonseed  meal  easily  takes  second  place 
and,  owing  to  its  superiority  in  the  sandy  soil,  dried  blood  takes 
third  place.  Considering  the  superiority  of  the  fish  guano  to 
the  dried  blood  in  the  other  soils,  however,  it  is  probably  fairer 
to  adjudge  dried  blood  and  fish  guano  of  equal  availability  from 
the  point  of  view  of  the  transformation  of  their  nitrogen  into 
ammonia  by  pure  cultures  of  ammonifying  bacteria. 


General  Discussion 

Several  of  the  facts  which  have  come  to  light  in  the  investiga- 
tions above  described  demand  a  word  of  comment  with  respect 
to  their  general  significance.  First  as  regards  the  relative 
efficiencies  at  ammonification  of  the  different  organisms  tested, 
we  find  that  there  is  marked  variation.  Indeed  it  is  difficult  to 
find  an  organism  among  the  fifteen  tested  which  consistently 
stands  as  the  best  ammonifier  regardless  of  the  soil  and  the 
ammonifiable  material  employed.  There  are,  however,  one  or  two 
organisms  which  nearly  approach  such  a  description.  In  other 
words,  it  appears  that,  viewing  ammonification  of  organic 
nitrogen  from  the  standpoint  of  pure  cultures,  every  organism 
will  do  best  with  a  definite  combination  of  soil  and  organic 
matter.  To  be  sure  there  are  some  organisms  of  those  tested,  even 
though  they  be  in  the  minority,  which  are  consistently  weak 
ammonifiers.  B.  icteroides  and  Ps.  fluorescens  serve  to  exemplify 
such. 

That  B.  mycoides  is  by  no  means  always  the  most  efficient  of 
ammonifying  bacteria  as  has  heretofore  been  believed  is  clearly 
indicated  above.  On  the  other  hand,  it  does  possess  and  manifest 
marked  superiority  in  certain  cases.  Thus,  for  example,  while 
showing  poor  or  mediocre  ammonifying  power  in  different  soils 
with  dried  blood,  tankage  and  fish  guano,  it  manifests  great  vigor 
in  the  case  of  cottonseed  meal  and  succeeds  in  making  the  record 
for  the  percentage  of  nitrogen  transformed  in  the  case  of  bat 
guano,  in  which  it  transforms  to  ammonia  36.06  per  cent  of  the 
nitrogen  present. 


168        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

Comparing  the  organisms  in  any  one  given  soil  as  a  medium 
we  find  some  interesting  facts.  In  the  sandy  soil,  for  example, 
with  dried  blood  B.  vulgaris  is  the  most  efficient  ammonifier, 
making  available  or  transforming  into  ammonia  24.36  per  cent 
of  the  nitrogen  present.  With  tankage,  B.  mesentericus  shows 
the  highest  efficiency,  transforming  32.52  per  cent  of  the  nitrogen 
present  into  ammonia.  With  cottonseed  meal,  B.  tumescens  is 
paramount,  yielding  an  amount  of  ammonia  equivalent  to  24.30 
per  cent  of  the  amount  of  nitrogen  present.  With  fish  guano, 
B.  vulgaris  again  manifests  its  superiority  over  the  other  organ- 
isms by  changing  13.09  per  cent  of  the  nitrogen  present  into 
ammonia.  When  bat  guano  is  used,  B.  mycoides  stands  distinctly 
superior  to  all  others,  as  above  shown,  by  transforming  36.06  per 
cent  of  the  nitrogen  present  into  ammonia.  In  the  case  of  sheep 
and  goat  manure  there  is  but  one  efficient  organism  and  that  is 
B.  megatherium,  which  transforms  24.97  per  cent  of  the  nitrogen 
present  into  ammonia.  Lastly  when  peptone  is  used  Sarcina 
lutea  stands  pre-eminent,  and  when  all  materials  are  compared, 
regardless  of  whether  they  were  used  in  all  soils  or  not,  the  last- 
named  organism  makes  the  record  for  availability  by  transform- 
ing 41.98  per  cent  of  the  nitrogen  present  into  ammonia.  There 
are  thus  six  organisms  out  of  the  fifteen  which  make  records  in 
one  and  the  same  soil  but  with  different  forms  of  organic  matter. 
One  of  the  six  stands  superior  in  the  cases  of  two  nitrogenous 
materials,  namely  dried  blood  and  fish  guano,  and  that  is 
B.  vulgaris. 

Comparing  the  same  organisms  with  the  same  nitrogenous 
materials,  above  used,  except  the  last  three  named,  in  the  clay- 
loam  soil,  we  find  that  with  dried  blood  B.  proteus  vulgaris  is 
most  efficient,  transforming  6.9  per  cent  of  the  nitrogen  present 
into  ammonia.  With  tankage,  B.  tumescens  is  pre-eminent  and 
transforms  12.74  per  cent  of  the  nitrogen  present  into  ammonia. 
With  cottonseed  meal,  Streptothrix,  sp.,  is  superior,  transforming 
12.84  per  cent  of  the  nitrogen  present  into  ammonia;  and  lastly 
with  fish  guano,  B.  megatherium  is  again  pre-eminent,  trans- 
forming 11.66  per  cent  of  the  nitrogen  present  into  ammonia. 
We  see  again,  therefore,  that  in  one  and  the  same  soil,  with  four 
different  materials,  four  different  organisms  make  records  for 


1914]        Lipman-Burgess :  Ammonification  in  Sails  by  Pure  Cultures  169 

availability.  To  be  sure,  two  of  these  organisms  have  shown 
themselves  superior  to  all  others  in  the  sandy  soil  but  with 
different  materials. 

In  the  case  of  the  clay-adobe  soil  we  find  that  with  dried 
blood  B.  tumescens  stands  at  the  top  of  the  list  and  transforms 
9.41  per  cent  of  the  nitrogen  present  into  ammonia.  With  tank- 
age in  the  same  soil,  Sarcina  lutea  is  the  most  efficient  ammonifier 
and  transforms  12.44  per  cent  of  the  nitrogen  present  into 
ammonia.  With  cottonseed  meal,  B.  tumescens  is  again  superior 
to  all  others  and  transforms  18.20  per  cent  of  the  nitrogen  present 
into  ammonia ;  and  finally  with  fish  guano  B.  vulgaris  again 
assumes  the  ascendency  and  transforms  8.64  per  cent  of  the 
nitrogen  present  into  ammonia.  B.  tumescens  is  very  nearly  as 
efficient  in  this  latter  case  as  B.  vulgaris.  We  have  thus  seen 
that  organisms  which  in  all  cases  have  shown  their  superiority 
in  other  soils  are  also  very  efficient  in  the  clay-adobe  soil. 

It  is  clear,  therefore,  that  only  about  half  of  the  fifteen 
organisms  tested  show  greatest  efficiency  in  some  soil  or  with 
some  form  of  organic  matter.  Scrutinizing  more  closely  the 
efficiencies  of  each  of  these,  we  must  concede  to  B.  tumescens  the 
paramount  place  among  them,  for  it  has  stood  pre-eminent,  in 
five  combinations  of  soil  and  fertilizer,  out  of  fifteen,  and  has  in 
addition  been  close  to  first  place  in  several  other  instances. 

Comparing  our  findings  with  those  of  Marchal,  the  following 
critical  statements  must  be  made.  First,  that  results  of  solution 
cultures  are  no  criterion  as  to  results  to  be  obtained  in  soils. 
Secondly,  that  no  two  forms  of  organic  nitrogen  are  attacked  and 
ammonified  with  the  same  vigor  by  any  one  organism.  Thirdly, 
that  different  soils  will  modify  an  organism 's  power  to  ammonify 
any  one  given  form  of  nitrogen  very  markedly,  so  that  it  may  be 
efficient  in  one  case  and  feeble  in  another.  Fourthly,  that  the 
ammonifying  efficiency  of  organisms  is  greater  in  sandy  soil,  and 
possibly  in  others,  than  in  solutions,  for  we  have  obtained  a 
transformation  of  41.98  per  cent  of  peptone  nitrogen  and  36.06 
per  cent  of  bat  guano  nitrogen  into  ammonia  by  Sarcina  lutea 
and  B.  mycoides  respectively  in  twelve  days  at  temperatures 
between  27°  C  and  30°  C,  while  Marchal  only  obtained  similar 
transformations  in  thirty  days  at  30°  C  in  albumin  solutions. 


170        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

Owing  to  the  general  distribution  of  the  efficient  ammonifying 
organisms  above  described  in  most  soils,  it  is  extremely  improb- 
able, to  judge  from  our  results,  that  we  may  look  forward 
to  a  profitable  form  of  inoculation  of  soils  with  ammonifying 
bacteria.  The  choice,  however,  of  the  form  of  nitrogenous  fer- 
tilizer for  a  soil,  which  will  be  most  readily  made  available,  when 
available  nitrogen  is  needed,  may  indeed  be  something  of  much 
greater  practical  significance,  as  our  data  would  appear  to 
indicate.  Particularly  emphatic  are  our  results  in  illustrating 
that,  at  least  so  far  as  pure  cultures  are  concerned,  and  as  also 
partly  shown  by  J.  G.  Lipman3  and  others,  with  mixed  cultures, 
prevalent  ideas  with  respect  to  the  relative  availability  of  common 
nitrogenous  fertilizers  are  incorrect.  Both  tankage  and  cotton- 
seed meal,  and,  in  some  cases,  fish  guano,  show  higher  avail- 
abilities than  dried  blood,  which  we  have  always  been  in  the 
habit  of  regarding  as  the  most  available  of  organic  nitrogenous 
fertilizers  (based  only  on  vegetation  tests). 

Nor  do  we  argue  too  far  afield  from  our  subject  when  we 
make  the  remarks  just  preceding.  We  recognize  fully  that 
availability  as  measured  by  ammonification  does  not  necessarily 
denote  availability  as  measured  by  nitrification  nor  by  assimila- 
tion of  nitrogen  by  plants.  We  cannot,  however,  help  noting, 
also,  that  a  persistent  preference  exists  among  practical  orchard- 
ists  in  this  state  for  tankage  as  against  dried  bood  and  we 
therefore  feel  that,  judged  by  other  critera,  similar  conclusions, 
must  be  drawn  to  those  adduced  from  our  experiments  with  pure 
cultures  of  ammonifying  bacteria. 

We  have  decided,  however,  to  go  much  further  in  these  experi- 
ments and  are  now  prosecuting  more  elaborate  investigations  on 
nitrogen  transformation  not  only  to  ammonia  but  also  to  nitrates. 
In  these  studies  we  shall  deal  with  the  soil  flora  as  existing  in  a 
large  variety  of  soil  types  freshly  collected  from  the  field  for  our 
purposes.  Moreover,  we  shall  employ  the  raw,  unsifted  fertilizer 
material  just  as  it  is  used  by  the  farmer.  From  these  investiga- 
tions we  hope  to  glean  much  more  evidence  which  will  be  of 
great  practical  significance,  and,  like  the  results  above  described, 
also  of  marked  scientific  interest. 


1914]        Lipman-Burgess :  Ammonifieation  in  Soils  by  Pure  Cultures  171 

Summary 
Results  are  above  given  which  deal  with : 

1.  The  marked  differences  in  ammonifying  efficiency  of  fifteen 
organisms  in  pure  culture. 

2.  The  soil  was  used  as  a  medium  and  three  types  employed, 
sandy,  clay  loam  and  clay  adobe. 

3.  Four  common  fertilizers  as  sources  of  nitrogen  were  used, 
in  all  soils,  and  peptone,  bat  guano,  and  sheep  and  goat  manure 
employed  only  in  the  sandy  soil,  besides.  The  four  fertilizers 
were  dried  blood,  tankage,  cottonseed  meal  and  fish  guano. 

4.  The  nature  of  the  soil,  as  well  as  the  nature  of  the  nitro- 
genous material,  markedly  modify  an  organism's  ammonifying 
power. 

5.  There  is  no  regularity  in  these  variations  and  they  cannot 
be  foretold. 

6.  While  it  is  difficult  to  make  an  exact  decision,  B.  tumescens 
appears,  on  the  whole,  to  have  been  the  most  efficient  organism 
above  tested. 

7.  The  highest  efficiency  in  a  single  culture  with  a  fertilizer 
was  shown  by  B.  mycoides,  which  transformed  in  twelve  days  at 
27°  to  30°  C,  36.06  per  cent  of  the  nitrogen  in  bat  guano  into 
ammonia. 

8.  The  highest  efficiency  in  a  single  culture  with  peptone  was 
shown  by  Sarcina  lutea,  which  transformed  41.98  per  cent  of  the 
nitrogen  present  into  ammonia  under  similar  conditions. 

9.  A  comparison  of  availability  of  nitrogenous  fertilizers  with 
ammonifiability  as  a  criterion,  according  to  our  experiments, 
markedly  changes  the  positions  of  tankage,  fish  guano,  and 
cottonseed  meal  with  respect  to  dried  blood,  showing  them  in 
most  or  in  many  cases  to  be  superior  to  the  latter. 

10.  One  half  of  the  number  of  organisms  tested  is  far  superior 
to  the  other  half  in  ammonifying  ability. 

11.  Interesting  comparisons  with  Marchal's  work  are  made. 

12.  B.  icteroides  has  shown  itself  throughout  to  possess  but 
little  ammonifying  efficiency. 


172        University  of  California  Publications  in  Agricultural  Sciences      [Vol.  1 

13.  Experiments  are  following  those  above  described  to  make 
the  latter  more  complete.  Elaborate  tests  are  being  made  with 
many  soil  types  using  the  original  soil 's  mixed  flora  from  freshly 
collected  samples  in  the  field.  Not  only  ammonifying  powers  of 
these  mixed  flora  in  the  different  soil  types  will  be  studied,  but 
also  the  corresponding  nitrifying  powers.  A  large  variety  of 
nitrogenous  fertilizers  as  employed  in  practice  will  be  tested  here. 


REFERENCES  OF  MORE  OR  LESS  DIRECT  PERTINENCE 

i  Bull.  Acad.  Roy.  Soc.  Belg.,  3  ser.,  vol.  25,  p.  727. 
-  Voorhees,  Fertilizers,  Macmillan  Co.,  1898. 

3  N.  J.  Sta.  Bulletin,  no.  246. 

4  Ann.  Sci.  Agron.,  vol.  19,  p.  209. 
s  Rpt.  Del.  Exp.  Sta.,  1899,  p.  76. 

e  Cent,  fur  Bakt.,  2le  Abt.,  vol.  20,  p.  322. 
7  Ibid.,  vol.  15,  p.  433. 
s  Ibid.,  vol.  29,  p.  238. 
o  Ibid.,  vol.  31,  p.  49. 


